This is generally in the field of production of polyhydroxyalkanoates by genetic engineering of bacterial enzymes.
Numerous microorganisms have the ability to accumulate intracellular reserves of poly [(R)-3-hydroxyalkanoate] (PHA) polymers. PHAs are biodegradable and biocompatible thermoplastic materials with a broad range of industrial and biomedical applications (Williams and Peoples, 1996, CHEMTECH 26: 38-44). PHAs can be produced using a number of different fermentation process and recovered using a range of extraction techniques (reviewed by Braunegg et al. 1998, J. Biotechnol. 65: 127-161; Choi and Lee, 1999). Plant crops are also being genetically engineered to produce these polymers offering a cost structure in line with the vegetable oils and direct price competitiveness with petroleum-based polymers (Williams and Peoples 1996, CHEMTECH 26:38-44; Poirier, Y. 1999, Plant Biotechnology pp. 181-185). PHAs are formed by the action of a PHA synthase enzyme. As the polymer chains grow, they form insoluble granules. The PHAs can then be recovered and then converted into chemicals or converted into chemicals during the recovery process (Martin et al. PCT WO 97/15681). Therefore, in addition to their utility as polymers, the PHAs represent a unique mechanism for storing new chemistries in both microbial and plant crop systems.
PHA copolymers containing 3-hydroxyvalerate (3HV), especially PHBV, have been described extensively. Many wild type microorganisms are capable of producing 3HV-containing PHAs. PHBV has been produced commercially using Ralstonia eutropha (formerly Alcaligenes eutrophus) from glucose and propionate and from glucose and isobutyrate (U.S. Pat. No. 4,477,654 to Holmes et al.). A number of other microorganisms and processes are known to those skilled in the art (Braunegg et al. 1998, Journal of Biotechnology 65: 127-161). Poly(3HV) homopolymer has been produced using Chromobacterium violaceum from valerate (Steinbuchel et al., 1993, Appl. Microbiol. Biotechnol. 39:443-449). PHAs containing 3HV units have also been synthesized using recombinant microorganisms. Escherichia coli harboring the R. eutropha PHA biosynthesis genes has been used to produce PHBV from glucose and either propionate or valerate (Slater et al., 1992, Appl. Environ. Microbiol. 58:1089-1094) and from glucose and either valine or threonine (Eschenlauer et al., 1996, Int. J. Biol. Macromol. 19:121-130). Klebsiella oxytoca harboring the R. eutropha PHA biosynthesis genes has been used to produce PHBV from glucose and propionate (Zhang et al., 1994, Appl. Environ. Microbiol. 60:1198-1205). R. eutropha harboring the PHA synthase gene of Aeromonas caviae was used to produce poly(3HV-co-3HB-co-3HHp) from alkanoic acids of odd carbon numbers (Fukui et al., 1997, Biotechnol. Lett. 19:1093-1097).
PHA copolymers containing 3-hydroxypropionate units have also been described. Holmes et al. (U.S. Pat. No. 4,477,654) used R. eutropha to synthesize poly(3HP-co-3HB-co-3HV) from glucose and either 3-chloropropionate or acrylate. Doi et al. (1990, in E. A. Dawes (ed.), Novel Biodegadable Microbial Polymers, Kluwer Academic Publishers, the Netherlands, pp. 37-48) used R. eutropha to synthesize poly(3HP-co-3HB) from 3-hydroxypropionate, 1,5-pentanediol, 1,7-heptanediol, or 1,9-nonanediol. Hiramitsu and Doi (1993, Polymer 34:4782-4786) used Alcaligenes latus to synthesize poly(3HP-co-3HB) from sucrose and 3-hydroxypropionate. Shimamura et al. (1994, Macromolecules 27: 4429-4435) used A. latus to synthesize poly(3HP-co-3HB) from 3-hydroxypropionate and either 3-hydroxybutyrate or sucrose. The highest level of 3-hydroxypropionate incorporated into these copolymers was 88 mol % (Shimamura et al., 1994, ibid.). No recombinant 3HP containing PHA producers have been described in the art.
It is economically desirable to be able to produce these polymers in transgenic crop species. Methods for production of plants have been described in U.S. Pat. No. 5,245,023 and U.S. Pat. No. 5,250,430; U.S. Pat. No. 5,502,273; U.S. Pat. No. 5,534,432; U.S. Pat. No. 5,602,321; U.S. Pat. No. 5,610,041; U.S. Pat. No. 5,650,555: U.S. Pat. No. 5,663,063; WO 9100917, WO 9219747, WO 9302187, WO 9302194 and WO 9412014, Poirier et.al., 1992, Science 256; 520-523, Williams and Peoples, 1996, Chemtech 26, 38-44, the teachings of which are incorporated by reference herein). In order to achieve this goal, it is necessary to transfer a gene, or genes in the case of a PHA polymerase with more than one subunit, encoding a PHA polymerase from a microorganism into plant cells and obtain the appropriate level of production of the PHA polymerase enzyme. In addition it may be necessary to provide additional PHA biosynthetic genes, e.g. a ketoacyl-CoA thiolase, an acetoacetyl-CoA reductase gene, a 4-hydroxybutyryl-CoA transferase gene or other genes encoding enzymes required to synthesize the substrates for the PHA polymerase enzymes. In many cases, it is desirable to control the expression in different plant tissues or organelles. Methods for controlling expression in plant tissues or organelles are known to those skilled in the art (Gasser and Fraley, 1989, Science 244; 1293-1299; Gene Transfer to Plants,1995, Potrykus, I. and Spangenberg, G. eds. Springer-Verlag Berlin Heidelberg New York. and "Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins", 1996, Owen, M. R. L. and Pen, J. Eds. John Wiley & Sons Ltd. England, incorporated herein by reference).
Although methods for production of a variety of different copolymers in bacterial fermentation systems are known, and production of PHAs in plants has been achieved, the range of copolymers possible in bacteria has not been achieved in plants. It would be advantageous to be able to produce different copolymers in transgenic plants, and to have more options with regard to the substrates to be utilized by the transgenic plants.
It is therefore an object of the present invention to provide methods and reagents for production of PHAs in plants.
It is a further object of the present invention to provide methods and reagents for production of PHAs using simple sugars and alcohols as substrates.
It is still another object of the present invention to provide methods and reagents for production of copolymers other than PHB and PHVB.